Arsenic is a prevalent environmental pollutant that poses a significant health risk worldwide. There is now compelling evidence that chronic exposure to arsenic is carcinogenic and that such exposure results in cancers of the skin and various internal organs. However, arsenic is not directly mutagenic and the oncogenic mechanism of action remains largely unclear. The overall goal of the experiments described in this proposal is to elucidate growth regulatory pathways effected by arsenic treatment in cultured human keratinocytes with a view towards understanding mechanisms underlying the oncogenic processes induced by long term arsenic exposure in humans. Towards this end we will use cDNA microarray analyses to identify growth regulatory genes in human keratinocytes whose expression is modulated by exposure of the cells to arsenite at low (submicromolar) levels. The microarray data will be subjected to cluster analyses to discern groups of genes that fall into groups based upon the manner in which their expression is modulated by arsenic treatment, in particular genes that show similar up- or down- regulation profiles as functions of time and dose. We will also carry out a series of experiments to examine the role of arsenite-mediated activation of D-type cyclins as effectors of premature exit from the GI: i) We will examine expression levels and phosphorylation status of intermediates in a receptor-mediated pathway that regulates expression of cyclin D1 via beta-catenin in order to determine how arsenic might interact with the beta-catenin pathway to effect changes in the steady-state levels of cyclin D and, 2) We will use a new technique based on transduction of a peptide containing the HIV TAT transduction protein fused to a small segment of the p16 INK-4a to specifically block the cyclin D-cdk4/cdk6 kinase. By observing changes in the length of the G1 phase as function of kinase inhibition (Rb phosphorylation) we can determine whether arsenic directly enhances G1 exit via cyclin D-cdk4/cdk 6 kinase activity.